Display technologies have played an important role in the isolation of specific high-affinity binding proteins for diagnostic and therapeutic applications in a vast number of disorders and diseases. These technologies extend into the broad field of antibody engineering, synthetic enzymes, proteomics, and cell-free protein synthesis. Biomolecular display technologies, which allow the construction of a large pool of modularly coded biomolecules, their display for property selection, and rapid characterisation (decoding) of their structures, are particularly useful for accessing and analyzing protein diversity on a large scale. Recently, in vitro display technologies have come to prominence due to the isolation of antibodies by phage display, ribosome display and microbial display, which have now become mainstream antibody and protein engineering platforms. However, microbial expression and display systems suffer from limitations in particular for the expression of large, dimeric vertebrate proteins, like antibodies. This is due to the general inability to express full-length antibodies in such expression systems, which requires the display of engineered antibody fragments, but also due to the lack of glycosylation, absence of chaperone proteins, lack of subcellular compartments and eukaryotic cell specific protein trafficking, that individually and collectively result in protein folding artefacts in microbially expressed mammalian proteins. Recently, in vitro display methods have also been developed employing eukaryotic host cells, including yeast, plants and mammalian cells. Yeast and plant cell expression systems also suffer from a lack of glycosylation and specific vertebrate and mammalian cell-specific chaperones, so that the same limitations with regard to protein folding apply for the expression of vertebrate proteins in such systems. Expression, proper protein folding and posttranslational modification of large recombinant proteins, like antibodies, can only be expected to occur with reasonable efficiency and quality in vertebrate expression systems, ideally expressing proteins in the phylogenetically most closely related cell system.
Therefore, therapeutically interesting proteins, like antibodies from rodents or humans, are ideally expressed in rodent or human cells, and it is not surprising that only expression systems from such species are approved by regulatory authorities for the production of clinically-grade full-length therapeutic antibodies. However, vertebrate and mammalian cell based expression systems are laborious, require long-time frames to establish stably producing cell lines and clones, and an efficient and controlled genetic modification of such cells is often not trivial and therefore makes these systems less attractive for screening and display methods. For instance, DNA transfection methods cannot be controlled for the number of DNA constructs that are either transiently or stably incorporated into transfected cells, which precludes clonal expression of protein libraries and therefore a clean gene to phenotype screen. The alternative viral systems either lack a proper control of clonal expression, a stable maintenance of the genetic constructs, and/or suffer from the fact that such systems often cause cytopathic effects in the target cells (e.g. vaccinia virus expression), such that protein clones either cannot be displayed and/or sequentially enriched for a particular phenotype, like e.g. specific binding to an antigen.
It is thus an object of the present invention to provide a method that clearly overcomes all of the above-mentioned limitations and drawbacks of prior art prokaryotic and eukaryotic gene expression and selection systems. The method according to the invention utilises stable retroviral expression of binding proteins such as, in particular, antibodies in mammalian cells, in particular B lymphocytic cell lines, such that stable and preferably clonal expression of antibody proteins is achieved in the presence of proper glycosylation, chaperone proteins and protein trafficking, ensuring proper protein folding and allowing efficient and, if desired, repeated screening for antigen-binding antibody clones. Since the preferred embodiment of the method according to the invention is based on the retroviral expression of antibodies or fragments thereof in precursor lymphocytes the technology disclosed herein is termed ‘Retrocyte Display’ (for retroviral preB lymphocyte display).